Autofluorescence Lifetime Imaging

Michael G. Nichols, Kristina Ward, Lyandysha V. Zholudeva, Heather Jensen Smith, Richard J. Hallworth

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations


Following a light-induced excitation, the fluorescence lifetime is the average residence time that a fluorophore will spend in its excited electronic state prior to returning to the ground state (Figure 4.1). Since the excited state is energetically unstable, this time (usually in the range of picoseconds to microseconds) is sensitive to a number of intramolecular interactions, defined by the fluorophore’s structure and the surrounding microenvironment. Such sensitivity makes the fluorescence lifetime an important observable for identifying a molecule and its conformational states (e.g., folding and ligand binding) and sensing its microenvironment (e.g., pH, temperature, and collisional encounters with other molecules or surfaces). While such interactions will also affect the spectral signature of the molecule, these changes are usually minor and often difficult to efficiently resolve due to their characteristically broad emission spectra. In contrast, changes in the fluorescence lifetime can be very pronounced (as much as 20-fold in some cases). In addition, the fluorescence lifetime does not depend on the concentration of the fluorophore under most experimental conditions, which makes fluorescence lifetime probing less sensitive to artifacts produced by photobleaching and scattering. These advantages are utilized in fluorescence lifetime imaging microscopy (FLIM) techniques-a number of methods complimentary to conventional fluorescence intensity measurements, such as wide-field and confocal microscopy (Bastiaens and Squire 1999; Gadella et al. 1993; Lakowicz 2006; Lakowicz and Berndt 1991; Levitt et al. 2009; Periasamy and Clegg 2009; Wang et al. 1989, 1992). While single-point fluorescence lifetime measurements of molecules in different environments have been around for almost a century, the first spatially resolved FLIM image was acquired 20 years ago (Berezin and Achilefu 2010; Wang et al. 1989). Since then, the applications of FLIM techniques have grown exponentially in both basic and applied research. In particular, FLIM of intrinsically fluorescent natural biomarkers of cellular metabolism, such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), has been applied to clinical research and diagnostics. Over the last two decades, FLIM technology has matured from the design-and-build-it-yourself days of the 1990s into commercially available FLIM upgrade kits for most standard fluorescence microscopes.

Original languageEnglish (US)
Title of host publicationNatural Biomarkers for Cellular Metabolism
Subtitle of host publicationBiology, Techniques, and Applications
PublisherCRC Press
Number of pages29
ISBN (Electronic)9781466509993
StatePublished - Jan 1 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Engineering(all)
  • Physics and Astronomy(all)


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